The identification and characterization of the molecular pathways involved in the early steps of cardiac cell proliferation are absolutely critical to understanding the pathologies and treatment of congenital heart disease. However, to date the early molecular pathways that control the progression of the embryonic cardiac cell cycle remain largely unknown. To address these issues, we cloned and characterized the Xenopus T-box gene Tbx5, the gene mutated in the human congenital heart disease Holt Oram syndrome (HOS). We have shown that TBX5 is both necessary and sufficient in vivo for the cardiac G1/S-transition of the cell cycle. From these and other studies, we hypothesize that TBX5 functions to maintain proliferation of cardiac progenitor populations. Xenopus offers an unparalleled opportunity to address this hypothesis due to the access of unlimited embryonic cardiac tissue, the development of cardiac explant assays, the availability EGFP-transgene reporter frogs that mark gene expression domains and mark specific phases of the cell cycle in living cardiac tissues, and our recent description of an extensive panel of antibodies that mark cell cycle components in the developing Xenopus heart. Here we propose to use TBX5 as a starting point to elucidate the molecular networks which control the proliferation of cardiac progenitor cells. This will be accomplished by determining if TBX5 directly regulates cyclin D1 and cyclin E2 in the embryonic heart, characterizing the biological significance of the SIN3B-TBX5 protein-protein interaction in cardiac cell cycle regulation, and through the identification of the endogenous cardiac mitogens which function through TBX5 to regulate the G1 to S transition of the cardiac cell cycle. PUBLIC HEALTH RELEVANCE The ability to isolate and propagate cell populations that can differentiate into cardiomyocytes in vivo offers the opportunity to treat a wide range of cardiac diseases. This proposal focuses on the characterization of the transcription factor TBX5, the gene mutated in the congenital heart disease Holt Oram syndrome, and its endogenous role in cardiac proliferation. Our immediate goal is to define and characterize the molecular pathways by which TBX5 functions with the overall goal, to use TBX5 as a starting point in an effort to begin to elucidate the pathways and molecular networks which control the survival and proliferation of cardiomyocyte progenitor cells.